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A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid.

ABSTRACT: Using mass spectrometry we have detected both assembly intermediates and the final product, the T=3 viral capsid, during reassembly of the RNA bacteriophage MS2. Assembly is only efficient when both types of quasiequivalent coat protein dimer seen in the final capsid are present in solution. NMR experiments confirm that interconversion of these conformers is allosterically regulated by sequence-specific binding of a short RNA stem-loop. Isotope pulse-chase experiments confirm that all intermediates observed are competent for further coat protein addition, i.e., they are all on the pathway to capsid formation, and that the unit of capsid growth is a coat protein dimer. The major intermediate species are dominated by stoichiometries derived from formation of the particle threefold axis, implying that there is a defined pathway toward the T=3 shell. These results provide the first experimental evidence for a detailed mechanistic explanation of the regulation of quasiequivalent capsid assembly. They suggest a direct role for the encapsidated RNA in assembly in vivo, which is consistent with the structure of the genomic RNA within wild-type phage.

SUBMITTER: Stockley PG

PROVIDER: S-EPMC7612263 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid.

Stockley Peter G PG Rolfsson Ottar O Thompson Gary S GS Basnak Gabriella G Francese Simona S Stonehouse Nicola J NJ Homans Steven W SW Ashcroft Alison E AE

Journal of molecular biology 20070315 2

Using mass spectrometry we have detected both assembly intermediates and the final product, the T=3 viral capsid, during reassembly of the RNA bacteriophage MS2. Assembly is only efficient when both types of quasiequivalent coat protein dimer seen in the final capsid are present in solution. NMR experiments confirm that interconversion of these conformers is allosterically regulated by sequence-specific binding of a short RNA stem-loop. Isotope pulse-chase experiments confirm that all intermedia ...[more]

PMID: 17434527

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Project description:The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP(2)) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T=3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qbeta known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T=3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP(2). We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5' or 3' end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T=3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T=3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.

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A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid.

Publications

A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid.

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